Nuclear receptors constitute a large superfamily of ligand-dependent and sequence-specific transcription factors. Members of this family influence transcription either directly, through specific binding to the promoters of target genes (see Evans, in Science 240:889-895 (1988)), or indirectly, via protein-protein interactions with other transcription factors (see, for example, Jonat et al., in Cell 62:1189-1204 (1990), Schuele et al., in Cell 62:1217-1226 (1990), and Yang-Yen et al., in Cell 62:1205-1215 (1990)). The nuclear receptor superfamily (also known in the art as the “steroid/thyroid hormone receptor superfamily”) includes receptors for a variety of hydrophobic ligands, including cortisol, aldosterone, estrogen, progesterone, testosterone, vitamin D3, thyroid hormone and retinoic acid, as well as a number of receptor-like molecules, termed “orphan receptors” for which the ligands remain unknown (see Evans, 1988, supra). These receptors all share a common structure indicative of divergence from an ancestral archetype.
Lipophilic hormones such as steroids, retinoic acid, thyroid hormone, and vitamin D3 control broad aspects of animal growth, development, and adult organ physiology. The effects of these hormones are mediated by a large superfamily of intracellular receptors that function as ligand-dependent and sequence-specific transcription factors. The non-steroidal nuclear receptors for thyroid hormone (TR), vitamin D3 (VDR), all-trans retinoic acid (RAR), and fatty acids and eicosanoids (PPAR) form heterodimers with the 9-cis retinoic acid receptor (RXR) that bind bipartite hormone-response elements (HREs) composed of directly repeated half sites related to the sequence AGGTCA (Mangelsdorf and Evans, Cell 83: 841-850, 1995). In contrast, the steroid receptors function as homodimers and bind to palindromic target sequences spaced by three nucleotides (Beato et al., Cell 83: 851-857, 1995). In addition to the known receptors, a large group of structurally-related “orphan” nuclear receptors has been described which possess obvious DNA and ligand binding domains, but lack identified ligands (Mangelsdorf et al., Cell 83:835-839, 1995; Enmark and Gustafsson, Mol. Endocrinol. 10:1293, 1996); and O'Malley and Conneely, Mol. Endocrinol. 6:1359, 1992)). Each has the potential to regulate a distinct endocrine signaling pathway.
It is widely viewed that the hormone response is a consequence of the release, from an endocrine gland, of a ligand that circulates through the blood, and coordinately regulates responses in target tissues by acting through specific nuclear receptors. Hormone responsiveness is dependent on the ability to rapidly clear ligand from the blood and the body so that, in the absence of a stimulus, target tissues return to a ground state. Hormonal homeostasis is thus achieved by the coordinated release and degradation of bioactive hormones. Steroid hormones and their many metabolites are primarily inactivated by reduction and oxidation in the liver. Since hundreds of adrenal steroids have been identified (e.g., dozens of each of the sex steroids (androgens, estrogens and progestins), 25-35 vitamin D metabolites, and likely hundreds of fatty acids, eicosanoids, hydroxyfats and related bioactive lipids), the problem of efficient ligand elimination is critical to physiologic homeostasis. In addition to the existence of a myriad of endogenous hormones, a similar diversity of ingested plant and animal steroids and bioactive xenobiotic compounds must also be degraded. Such compounds often are lipophilic and may accumulate to toxic levels unless they are metabolized to water-soluble products that can be readily excreted. Therefore, the efficient detoxification of harmful xenobiotics is essential to the survival of all organisms.
Selye first introduced the concept that exogenous steroids and pharmacologic substances may function to modulate the expression of enzymes that would protect against subsequent exposure to toxic xenobiotic substances (H. Selye, J. Pharm. Sci. 60:1-28, 1971). These compounds, which Selye called “catatoxic steroids,” are typified by the synthetic glucocorticoid antagonist, pregnenolone-16-carbonitrile (PCN). PCN, and a variety of xenobiotic steroids, induce the proliferation of hepatic endoplasmic reticulum and the expression of cytochrome P450 genes (Burger et al., Proc. Natl. Acad. Sci. (USA) 89:2145-2149, 1992; Gonzalez et al., Mol. Cell. Biol. 6:2969-2976, 1986; and Schuetz and Guzelian, J. Biol. Chem. 259:2007-2012, 1984). Cytochrome P450 (CYP) enzyme(s), present in the endoplasmic reticulum of livers, often catalyze the initial step in such detoxification pathways. P450's are crucial for the detoxification of most xenobiotics, including various environmental pollutants, procarcinogens, and drugs (for review see Denison M S and Whitlock Jr, J. Biol. Chem. 270:18175-18178, 1995). In addition, CYPs are also responsible for the reduction and oxidation of steroid hormones and their many metabolites.
One consequence of PCN treatment is the induction of nonspecific “protection” against subsequent exposure to such diverse xenobiotics as digitoxin, indomethacin, barbiturates, and steroids (Selye, supra, 1971). Furthermore, it is known that a variety of such compounds can activate P450 genes responsible for their detoxification or degradation (Fernandez-Salguero and Gonzalez, Pharmacogenetics 5:S123-128, 1995; Denison and Whitlock, supra 1995; O. Hankinson, Ann. Rev. Pharmacol. Toxicol. 35:307-340, 1995; and Rendic and Di Carlo, Drug Metab. Rev. 29:413-580, 1997). P450's constitute a superfamily; each form possesses an overlapping but distinct substrate specificity. Some P450 genes are expressed constitutively, while others, particularly those involved in xenobiotic metabolism, are inducible. In many cases, inducers are also substrates for the induced enzymes, therefore, P450 activities typically remain elevated only as needed. Among the CYP gene family members, the CYP3A isoenzyme is of particular significance from a medical perspective. The human CYP3A4 enzyme is involved in the metabolism of a large number of clinical drugs including antibiotics, antimycotics, glucocorticoids, and the statin class of the HMG-CoA reductase inhibitors (Maurel P, Ioannides C Ed. (CRC Press, Boca Raton, Fla.) pp. 241-270, 1996). Indeed, the drug-induced CYP3A4 activation constitutes the molecular basis for a number of important clinically known drug-drug interactions. CYP3A23 and CYP3A11 are rodent homologues of CYP3A4 in rat and mouse, respectively. Indeed, purified CYP3A11 (P450MDX-B) exhibited comparable activity to CYP3A1 (another rat CYP3A homologue, Halvorson, et al., Arch. Biochem. Biophys. 277:166-180, 1990) and CYP3A4 (Yamazaki, and Shimada, Arch. Biochem. Biophys., 346:161-169, 1996) for testosterone 6β-hydroxylation, which is thought to be one of the specific reactions for the CYP3A enzyme in rodents and primates (Matsunaga et al., 1998). The regions of the 5′ regulatory sequences of CYP3A23 and CYP3A11 share high homology, including multiple putative response elements (Toide et al., Arc. Biochem. Biophy. 338:43-49, 1997), indicating similar transcriptional regulatory mechanisms among these rodent CYP3A genes.
Although there are substantial structural and catalytic similarities among the various members of the CYP3A family across species lines, important differences exist in regulatory control of these genes (for review, see Gonzalez F J., Pharmacol. Ther. 45:1-138, 1990, and Nelson D R., Arch. Biochem. Biophy., 369:1-10, 1999). For example, a clear discrepancy between human and rodents is that the antibiotic RIF induces CYP3A4 in human liver (Watkins et al., N Engl J Med 338:916-917, 1985) but does not induce CYP3A23 in rats (Wrighton et al, Mol Parmacol 28:312-321, 1985) and CYP3A11 in mice (Schuetz et al., Proc Natl Acad Sci USA 93:4001-4005, 1996), respectively. On the other hand, the anti-glucocorticoid PCN, which induces CYP3A23 in rat liver (Wrighton et al, 1985), only weakly induces human CYP3A4 (Schuetz et al., Hepatology 18:1254-1262, 1993, Kocarek et al., Drug Metab Dispos 23:415-421, 1995, Blumberg et al, Genes Dev 12:3149-3155, 1998), and does not induce CYP3A6 (Dalet et al., DNA 7: 39-46, 1988), a rabbit homolog with a drug response specificity similar to CYP3A4 (Barwick et al, Mol Pharmacol 50:10-16, 1996). Given the widespread metabolic importance of CYP3A, it would be of great clinical benefit to find an appropriate animal model for use in developing a better understanding of the regulatory control and inter-individual heterogeneity in liver expression of CYP3A in humans.
While it appears that catatoxic compounds such as PCN regulate the expression of cytochrome P450s and other detoxifying enzymes, two lines of evidence argue that such regulation is independent of the classical steroid receptors. First, many of the most potent compounds (e.g., PCN, spironolactone, and cyproterone acetate) have been shown to be steroid receptor antagonists; whereas others (e.g., dexamethasone) are steroid receptor agonists (Burger, supra, 1992). Second, the nonspecific protective response remains after bilateral adrenalectomy (and presumably in the absence of adrenal steroids), but not after partial hepatectomy (Selye, supra, 1971).
Insight into the mechanism by which PCN exerts its catatoxic effects is provided by the demonstration that PCN induces the expression of CYP3A1 and CYP3A2, two closely related members of the P450 family of monooxygenases (see, for example, Elshourbagy and Guzelian in J. Biol. Chem. 255:1279 (1980); Heuman et al., in Mol. Pharmacol. 21:753 (1982); Hardwick et al., in J. Biol. Chem. 258:10182 (1983); Scheutz and Guzelian in J. Biol. Chem. 259:2007 (1984); Scheutz et al., in J. Biol. Chem. 259:1999 (1984); and Gonzalez et al., in J. Biol. Chem. 260:7435 (1985)). The CYP3A hemoproteins display broad substrate specificity, hydroxylating a variety of xenobiotics (e.g., cyclosporin, warfarin and erythromycin), as well as endogenous steroids (e.g., cortisol, progesterone, testosterone and DHEA-sulfate. See, for example, Nebert and Gonzalez in Ann. Rev. Biochem. 56:945 (1987) and Juchau in Life Sci. 47:2385 (1990)). A PCN response element (which is highly conserved in the CYP3A2 gene promoter) has since been identified in subsequent studies with the cloned CYP3A1 gene promoter (see Miyata et al., in Archives Biochem. Biophysics 318:71 (1995) and Quattrochi et al., in J. Biol. Chem. 270:28917 (1995)). This response element comprises a direct repeat of two copies of the nuclear receptor half-site consensus sequence AGTTCA.
In addition to inducing CYP3A gene expression, PCN has also been shown to have marked effects on hepatic cholesterol homeostasis. These effects include significant decreases in the levels of HMG-CoA reductase and cholesterol 7a-hydroxylase gene expression, with associated reductions in sterol biosynthesis and bile acid secretion. PCN has also been reported to enhance the formation of cholesterol esters and the hypersecretion of cholesterol into the bile. Thus, PCN affects key aspects of cholesterol metabolism, including its biosynthesis, storage and secretion.
Activation of orphan nuclear receptor(s) by catatoxic steroids provides a possible mechanism for the induction of xenobiotic metabolizing enzymes by compounds that do not activate known steroid receptors. Because such enzymes are activated by high (pharmacological) doses of xenobiotic and natural steroids, such a “sensor” would be expected to be a broad-specificity, low-affinity receptor. Such receptors could be activated not only by endogenous steroids and metabolites but also by exogenous compounds such as phytosteroids, xenobiotics and pharmacologic inducers. Indeed, it is known that a variety of such compounds can activate P450 genes responsible for their detoxification or degradation (see, for example, Fernandez-Salguero and Gonzalez in Pharmacogenetics 5:S123 (1995); Denison and Whitlock, Jr., supra, 1995); Hankinson in Ann. Rev. Pharmacol. Toxicol. 35:307 (1995); and Rendic and Di Carlo in Drug Metab. Rev. 29:413 (1997)).
In healthy individuals, steroid levels are tightly regulated, with increased catabolism of endogenous steroids being compensated by the pituitary releasing an increase of ACTH, which stimulates biosynthesis, and maintenance of plasma steroid levels. The increased catabolism is reflected by elevated urinary levels of steroid metabolites. Indeed, it is already known that treatment with rifampicin increases urinary metabolites, such as 6β-hydroxycortisol (Ohnhaus et al., Eur. J. Clin. Pharmacol. 36:39-46, 1989; and Watkins et al., J. Clin. Invest., 83:688-697, 1989), and bile acid metabolites, such as 6β-hydroxy hyocholic and 6α-hyodeoxycholic acids (Wietholtz et al., J. Hepatol, 24:713-718, 1996), while the plasma levels of many circulating steroids rise slightly due to increased synthesis (Lonning et al., J. Steroid Biochem. 33:631-635, 1989; Bammel et al., Eur. J. Clin. Pharmacol, 42:641-644, 1992; and Edwards et al., Lancet 2:548-551, 1974).
When synthetic steroids, such as prednisolone (McAllister et al., Br. Med. J. 286:923-925, 1983; and Lee et al., Eur. J. Clin. Pharmaco, 45:287-289, 1993) or 17α-ethynylestradiol (F. P. Guengerich, Life Sci., 47:1981-1988, 1990) are administered together with rifampicin, plasma levels are rapidly decreased due to enhanced urinary clearance. In some patients undergoing rifampicin therapy for tuberculosis, the increase in urinary steroid levels has led to misdiagnosis of Cushing's syndrome (Kyriazopoulou and Vagenakis, J. Clin. Endocrinol. Metab., 75:315-317, 1992; Zawawi et al., Ir. J. Med. Sci., 165:300-302, 1996; and Terzolo et al., Horm. Metab. Res., 27:148-150, 1995). In these patients, steroid production and clearance normalized when rifampicin was withdrawn. In patients with Addison's disease, who mostly lack the ability to synthesize adrenal steroids, rifampicin treatment leads to rapid depletion of endogenous and administered steroids. These documented clinical situations confirm that induction of CYP3A4 causes increased steroid catabolism (Kyriazopoulou et al., J. Clin. Endocrinol. Metab. 59:1204-1206, 1984; and Edwards, supra, 1974). However, the art is silent regarding the mechanism by which steroid metabolism is regulated in the body.
Although therapeutically administered steroids are beneficial in achieving therapeutic goals, such compounds can, in some cases, increase the overall level of steroids and xenobiotics above physiologically compatible levels in the subjects to whom they are administered. In other cases, the increased level of steroids and/or xenobiotics may linger in the body longer than is therapeutically required. In addition, some subjects are treated with combinations of steroids and xenobiotics that may be administered separately to treat different conditions, but which, in combination, have an additive, or even synergistic, effect known as a drug interaction. In such cases, the patient may be unaware when a physiologically incompatible level of steroids and xenobiotics has been reached, or when an otherwise therapeutic amount of a steroid becomes potentially dangerous due to combined effects of separately administered drugs.
Thiazolidinediones (TZDs) are a new class of oral antidiabetic agents, and have been identified to be the synthetic ligands for peroxisome proliferator-activated gamma (PPARγ) (for reviews, see Spiegelman, 1998, and Wilson and Wahli, 1997). Troglitazone is the first TZD introduced for clinical use. Although troglitazone is effective in reducing hyperglycemia, concern has been raised by several reports of severe hepatic dysfunction leading to hepatic failure in patients receiving the drug (Neuschwander-Tetri et al, 1998, Shibuya et al., 1998, and for a review, see Watkins and Whitcomb, 1998). The mechanism of the liver toxicity by TZDs remains largely unknown.
Accordingly, there is still a need in the art for the identification and characterization of broad specificity, low affinity receptors that participate in the mediation of the physiological effect(s) of steroids and xenobiotics, particularly when combinations of such compounds disrupt homeostasis or cause drug interaction.